Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. We propose to elucidate fine point structural details of inter-protein interactions that will define mechanisms for factor VIIIa-dependent catalytic rate enhancement of factor IXa and the down- regulation of its activity leading to dampening of the intrinsic factor Xase. Aim I studies mechanisms by which factor VIIIa modulates catalytic efficiency within factor Xase. We continue to study the role of the factor VIIIa A2 subunit as this subunit directly influences events at the active site of factor IXa. Use of novel recombinant A2 reagents will facilitate identification of critical factor IXa-interactive residues. A new focus of these studies now examines the role of the factor VIIIa A3C1C2 subunit in factor Xase formation. This aim is significant since this subunit provides the majority of the binding energy for the interaction of factor VIIIa with factor IXa. Our development of a baculovirus expression system yielding fully functional A3C1C2 subunit will be instrumental in addressing this aim. Factor VIIIa enhances substrate factor X binding in Xase and we will investigate the stabilization of a factor X-interactive site in the factor VIIIa A1 subunit by the A3C1C2 subunit. The role of positive charge potential in the A2 subunit in contributing to substrate turnover and product release will be evaluated using recombinant reagents. Aim II focuses on proteolytic inactivation of factor VIIIa and is based upon our recent studies showing exosite-dependence for cofactor inactivation by activated protein C (APC) and factor Xa, as well as an important contribution of sequences flanking the P1 Arg residues in APC-catalyzed inactivation of factor VIIIa. The molecular interactions leading to reactions of these enzymes responsible for factor VIIIa inactivation and consequent dampening of factor Xase remain poorly understood. We will probe these interactions using native and mutant factor VIIIa variants possessing alterations in putative exosite-interactive regions as well as with cleavage-resistant forms. A focal point is proteolysis at Arg336 in the A1 subunit, the predominant site cleaved by both APC and factor Xa, and elucidation of mechanisms for this catalytic event. These aims will be facilitated by use of novel reagents including recombinant protease forms, substrate comprised of highly purified factor VIII(a) chains/subunits, as well as variants possessing point mutations at residues of interest. We will accomplish these studies using high resolution techniques including fluorescence energy transfer and anisotropy, surface plasmon resonance and MALDI-TOF mass spectrometry. We anticipate our results will define mechanisms providing significant insights into cofactor-mediated catalysis and its regulation, as well as provide useful information for the design of superior therapeutics for the treatment of hemophilia A. PUBLIC HEALTH RELEVANCE: Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. In this application we will elucidate fine point structural details of inter-protein interactions that will define mechanisms for factor VIIIa cofactor function and the regulation of its activity in factor Xase.